O-Glycan analysis by CE-MS

One of the most important co- and post-translational modification of a protein is glycosylation (the attachment of a sugar moiety group, also known as glycans). This modification plays a vital role in various biological events such as cellular communications, their signaling process, immune system, protein folding and stability. Minor changes in the glycosylation pattern are related to various diseases such as cancer. As glycosylation is involved in almost all biological events, it is an ideal biomarker candidate. Glycosylation is divided in various types, where protein glycosylation is either N- or O-linked. The latter is the least studied of the two due to the lack of generic enzymes, their often complex structure, large heterogeneity and the variety in core structures. Moreover, glycans can exist in various isomeric structures, which cannot readily be distinguished by solely mass spectrometry (MS). The requirement for a separation technique capable of analyzing low-abundant species, highlights the significance of capillary electrophoresis (CE) in recent years, because of its high sensitivity, ability to separate isomers, and the linkage analysis. Until now, the N-glycan class is the main form being explored by CE-MS at a released level, leaving O-glycosylation relatively unexplored. This is mainly related to the separation mechanism of CE, as it is important that the analytes carry a charge, which in glycomic workflows is often introduced by labelling the reducing end of the glycan. As common release strategies (β elimination) for O-glycans make the reducing end unavailable for labelling, the analytes are more complicated to analyze by CE-MS. Just recently, a new releasing procedure was introduced that protects the reducing end, opening up a range of new opportunities. Notably, this method allows the sequential release of both N- and O-glycome from the same sample. Therefore, this study will focus on extending the glycoanalytical CE-MS portfolio by adding this layer and comparing it to the already developed LC-MS workflow. Both N- and O-glycan were sequentially released from total plasma and fetuin. After the release, an internal standard (DP7) was spiked to the sample, followed by a labeling procedure with either 2AB or GirP for LC-MS or CE-MS, respectively. The first experiments, revealed a successful release of N- and O-glycans from fetuin, as the most abundant forms could easily be detected at 30 ng by CE-MS. To ensure the highest sensitivity possible, further experiments will focus on further optimalization the workflow by critically evaluating the sample preparation step. Additionally, the workflow will be assessed on different type of biospecimens (e.g. plasma, tissue and cells). Eventually, this will allow us to gain a deeper understanding of the glycome and, eventually, decipher glycomic intra-tumoral heterogeneity in, for example, head and neck squamous cell carcinoma (HNSCC).